Infection of cells by herpes simplex virus type 1 (HSV-1) results in dramatic changes in RNA polymerase II (RNAP II) function, such that viral genes are highly transcribed in a regulated cascade, while host genes are transcriptionally silenced. This subversion of the host transcription machinery is mediated by a small set of viral regulatory proteins that are amenable to genetic and biochemical analysis. HSV-1 thus provides a powerful system to understand how RNAP II can be globally regulated by trans-acting factors. The fundamental hypothesis of this research is that HSV-1-encoded regulatory proteins induce physical and functional modifications to the RNAP II machinery which result in the preferential transcription of viral genes. To date we have identified three physical modifications to the RNAP II apparatus that are induced by HSV-1. These are: (a) the aberrant phosphorylation of the C-terminal domain (CTD) of RNAP II; (b) the loss of transcription factor TFIIE from the RNAP II holoenzyme, and (c) the association of several HSV-1 immediate-early proteins with RNAP II complexes. Our first specific aim is to use genetic and biochemical approaches to characterize the functions of the viral proteins ICP22 and UL13, focusing on their roles in CTD modification. We will engineer and analyze ICP22 mutants and an ICP22/UL13 mutant, and ask whether CTD modification is mediated by virion or newly-expressed UL13. We will also investigate whether ICP22 and UL13 are sufficient for RNAP II modification, and whether together they comprise a CTD kinase. Our second aim is to study the mechanisms and consequences of the HSV-1 -induced loss of TFIIE from the RNAP II holoenzyme. In vitro transcription assays will be used to test the functionality of the post-infection holoenzyme. We will also investigate if the post infection holoenzyme has an altered CTD kinase activity. Our third aim is to understand how the viral IE proteins inhibit cellular transcription. We will use transfection assays and mutant viruses to identify the IE proteins which mediate transcriptional repression, and we will study the mechanisms involved. In addition, we will investigate the hypothesis that HSV-1 inhibits the cellular transcriptional pathway by disrupting the normal association between the RNAP Il CTD and cellular cofactors that interact with it, specifically mRNA splicing factors and the transcriptional mediator complex. Together, the experiments outlined in this proposal will further our understanding of mechanisms by which trans-acting proteins can globally regulate transcription. This research will also help to elucidate the fundamental replication mechanisms of HSV-1 and other pathogenic herpesviruses.